Theranostics 2021; 11(14):6746-6765. doi:10.7150/thno.60701

Research Paper

A novel cell-penetrating peptide targeting calpain-cleavage of PSD-95 induced by excitotoxicity improves neurological outcome after stroke

Sara Ayuso-Dolado, Gema M. Esteban-Ortega*, Óscar G. Vidaurre*, Margarita Díaz-Guerra

Instituto de Investigaciones Biomédicas “Alberto Sols”, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid 28029, Spain.
*These authors contributed equally to this work.

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Ayuso-Dolado S, Esteban-Ortega GM, Vidaurre ÓG, Díaz-Guerra M. A novel cell-penetrating peptide targeting calpain-cleavage of PSD-95 induced by excitotoxicity improves neurological outcome after stroke. Theranostics 2021; 11(14):6746-6765. doi:10.7150/thno.60701. Available from

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Postsynaptic density protein-95 (PSD-95) is a multidomain protein critical to the assembly of signaling complexes at excitatory synapses, required for neuronal survival and function. However, calpain-processing challenges PSD-95 function after overactivation of excitatory glutamate receptors (excitotoxicity) in stroke, a leading cause of death, disability and dementia in need of efficient pharmacological treatments. A promising strategy is neuroprotection of the infarct penumbra, a potentially recoverable area, by promotion of survival signaling. Interference of PSD-95 processing induced by excitotoxicity might thus be a therapeutic target for stroke and other excitotoxicity-associated pathologies.

Methods: The nature and stability of PSD-95 calpain-fragments was analyzed using in vitro assays or excitotoxic conditions induced in rat primary neuronal cultures or a mouse model of stroke. We then sequenced PSD-95 cleavage-sites and rationally designed three cell-penetrating peptides (CPPs) containing these sequences. The peptides effects on PSD-95 stability and neuronal viability were investigated in the cultured neurons, subjected to acute or chronic excitotoxicity. We also analyzed the effect of one of these peptides in the mouse model of stroke by measuring infarct size and evaluating motor coordination and balance.

Results: Calpain cleaves three interdomain linker regions in PSD-95 and produces stable fragments corresponding to previously described PSD-95 supramodules (PDZ1-2 and P-S-G) as well as a truncated form SH3-GK. Peptide TP95414, containing the cleavage site in the PDZ3-SH3 linker, is able to interfere PSD-95 downregulation and reduces neuronal death by excitotoxicity. Additionally, TP95414 is delivered to mice cortex and, in a severe model of permanent ischemia, significantly improves the neurological outcome after brain damage.

Conclusions: Interference of excitotoxicity-induced PSD-95-processing with specific CPPs constitutes a novel and promising therapeutic approach for stroke treatment.

Keywords: cell-penetrating peptides, excitotoxicity, neuroprotection, PSD-95, stroke